RESEARCH ARTICLE Open Access Genome wide analysis of MicroRNA messenger RNA interactome in ex vivo gill filaments, Anguilla japonica Hoi Man Ng1†, Jeff Cheuk Hin Ho1†, Wenyan Nong2, Jerome Ho Lam Hui2[.]
Ng et al BMC Genomics (2020) 21:208 https://doi.org/10.1186/s12864-020-6630-0 RESEARCH ARTICLE Open Access Genome-wide analysis of MicroRNAmessenger RNA interactome in ex-vivo gill filaments, Anguilla japonica Hoi Man Ng1†, Jeff Cheuk Hin Ho1†, Wenyan Nong2, Jerome Ho Lam Hui2, Keng Po Lai3* and Chris Kong Chu Wong1* Abstract Background: Gills of euryhaline fishes possess great physiological and structural plasticity to adapt to large changes in external osmolality and to participate in ion uptake/excretion, which is essential for the re-establishment of fluid and electrolyte homeostasis The osmoregulatory plasticity of gills provides an excellent model to study the role of microRNAs (miRs) in adaptive osmotic responses The present study is to characterize an ex-vivo gill filament culture and using omics approach, to decipher the interaction between tonicity-responsive miRs and gene targets, in orchestrating the osmotic stress-induced responses Results: Ex-vivo gill filament culture was exposed to Leibovitz’s L-15 medium (300 mOsmol l− 1) or the medium with an adjusted osmolality of 600 mOsmol l− for 4, and 24 h Hypertonic responsive genes, including osmotic stress transcriptional factor, Na+/Cl−-taurine transporter, Na+/H+ exchange regulatory cofactor, cystic fibrosis transmembrane regulator, inward rectifying K+ channel, Na+/K+-ATPase, and calcium-transporting ATPase were significantly upregulated, while the hypo-osmotic gene, V-type proton ATPase was downregulated The data illustrated that the ex-vivo gill filament culture exhibited distinctive responses to hyperosmotic challenge In the hyperosmotic treatment, four key factors (i.e drosha RNase III endonuclease, exportin-5, dicer ribonuclease III and argonaute-2) involved in miR biogenesis were dysregulated (P < 0.05) Transcriptome and miR-sequencing of gill filament samples at and h were conducted and two downregulated miRs, miR-29b-3p and miR-200b-3p were identified An inhibition of miR-29b-3p and miR-200b-3p in primary gill cell culture led to an upregulation of 100 and 93 gene transcripts, respectively Commonly upregulated gene transcripts from the hyperosmotic experiments and miR-inhibition studies, were overlaid, in which two miR-29b-3p target-genes [Krueppel-like factor (klf4), Homeobox protein Meis2] and one miR-200b-3p target-gene (slc17a5) were identified Integrated miR-mRNA-omics analysis revealed the specific binding of miR-29b-3p on Klf4 and miR-200b-3p on slc17a5 The target-genes are known to regulate differentiation of gill ionocytes and cellular osmolality (Continued on next page) * Correspondence: kengplai@cityu.edu.hk; ckcwong@hkbu.edu.hk † Hoi Man Ng and Jeff Cheuk Hin Ho contributed equally to this work Guanxi Key Laboratory of Tumor Immunology and Microenvironmental Regulation, Guilin Medical University, Huan Cheng North 2nd Road 109, Guilin 541004, People’s Republic of China Croucher Institute for Environmental Sciences, Department of Biology, Hong Kong Baptist University, Kowloon Tong, HKSAR, Hong Kong Full list of author information is available at the end of the article © The Author(s) 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data Ng et al BMC Genomics (2020) 21:208 Page of 13 (Continued from previous page) Conclusions: In this study, we have characterized the hypo-osmoregulatory responses and unraveled the modulation of miR-biogenesis factors/the dysregulation of miRs, using ex-vivo gill filament culture MicroRNAmessenger RNA interactome analysis of miR-29b-3p and miR-200b-3p revealed the gene targets are essential for osmotic stress responses Keywords: Fish gill osmoregulation, Hyperosmotic stress, Japanese eel, Transcriptome, microRNA inhibitors Background The catadromous fish Japanese eels have a complex life cycle in freshwater and seawater environments The fish is euryhaline and actively engage physiological responses to oppose osmotic perturbations to stabilize body osmolality Fish gills therefore possess great structural and functional plasticity in response to variations of water osmolality, to support the process of fluid and electrolyte homeostasis [1, 2] In response to osmotic stress, a distinct suite of modifications in gill epithelia is activated for functional adaptation, which involves cell proliferation and differentiation, changes in the activities, expressions and trafficking of different ion transporters/ channels [3, 4] In the past decades, considerable numbers of reports identified the underlying molecular and physiological factors associated with ion-osmoregulatory functions of gills In recent, advances in the understanding of small non-coding RNAs such as microRNAs (miRs) on regulatory circuit in physiological and pathophysiological functions shed light on roles of miRs in osmotic stress responses In mammals, the involvement of miRs in fluid and electrolyte transport has become apparent A number of studies have illustrated the implications of miRs in ion transport functions For instances, a direct regulatory role of miR-9 in the expression of large-conductance calcium and voltage-activated potassium channels in neuronal ion transport in alcohol tolerance phenomenon in an adult rat model was identified [5] The involvement of miR-133a and miR-133b in primary culture of canine cardiomyocytes [6] and miR-1 [7] on potassium channels for cardiac arrhythmias in human samples and rat models were demonstrated Moreover, indirect regulatory roles of miRs (i.e miR-155, miR124 & miR-135a) on ion transport were reported via their inhibitory actions on receptors of electrolyte-regulated hormones, angiotensin-I receptor or mineralocorticoid receptor, in screening samples of patients with hypertension [8, 9] The expression level of the with-no-lysine kinase (wnk1, a regulator of electrolyte homeostasis) in mouse nephrons, was found to be regulated by miR-192 Its expression was modulated by physiological stimuli (i.e aldosterone or salts loading) [10] In renal medullary epithelial mIMCD3 cells, tonicity was also identified as a stimulus to regulate miR-200b and miR-717 expression, which inhibit the expression of the transcriptional factor, osmotic response element binding protein (orebp) [11] In comparison, studies of miRs in osmotic stress responses in fish are limited Using zebrafish embryos, the expression of the miR-8 family (miR-200) in ionocytes was found to inhibit the expression of Na+/K+-exchanger regulatory cofactor (nherf1) to impair cellular responses to osmotic stress [12] Using tilapia, decreased expression levels of miR-429 in gills under hyperosmotic stress was observed An in-vivo inhibitory effect of miR-429 on the expression of osmoregulatory transcription factor (ostf1) in gills [13] and an inhibitory action of miR-30c on the expression of renal hsp70 under hyperosmotic stress [14] were reported Moreover, differential expression patterns of miRs in gills of marbled eels, Anguilla marmorata adapted at different salinities [i.e freshwater (FW), brackish-water or seawater (SW)] were described [15] Japanese eels are euryhaline fish The osmoregulatory tissue - gills provides an excellent model to study role of miRs in the regulation on plasticity of adaptive osmotic responses in vertebrates The present study aimed to identify and characterize the involvement of miRs and messenger RNAs (mRNA) under osmotic perturbations In this report, we used both exvivo gill filament and primary gill cell culture models, accompanied with miR-, transcriptome-sequencing and miR inhibition, to identify tonicity-sensitive miRs and to characterize their expressions in gill filaments Results Hyperosmotic treatment induced differential expression of transcriptional Factor-1/regulators, ion channels/ transporters and miR biogenesis factors in ex-vivo gill filament culture Gill filaments of eels were ex-vivo cultured in isotonic (300 mOsmol l− 1) or hypertonic (600 mOsmol l− 1) L-15 medium (penicillin-streptomycin (PS), gentamycin, FBS) at 23 °C for 4, and 24 h Transcripts levels of ostf1, Na+/Cl−-taurine transporter (taut), Na+/H+ exchange regulatory cofactor (nherf1), cystic fibrosis transmembrane regulator (cftr), and inward rectifying K+ channel (kir), were significantly upregulated at hypertonic medium, in a time-dependent manner (Fig 1) Moreover, the adenosine triphosphatases (ATPases), including the subunits of Na+/K+-ATPase [ATPase Na+/K+ transporting subunit α1 (at1α1), ATPase Na+/K+ transporting Ng et al BMC Genomics (2020) 21:208 Page of 13 Fig Hypertonic stress induces differential gene expression of osmotic stress transcriptional factor and seawater ion transporter Eel gill filaments were challenged in hyperosmotic (Hyper: 600 mOsmol l− 1) media for 4, and 24 h Control (Ctrl: 300 mOsmol l− 1) or hypertonic-treated gill filaments were subsequently processed for analysis of tonicity-responsive gene expression by quantitative SYBR Green Real-Time PCR (qRT-PCR) Differential gene expression of osmotic-stress transcriptional factor (ostf1), Na+/K+ exchange regulatory cofactor (nherf1), with-no-lysine kinase (wnk1), inward rectifying K+ channel (kir), cystic fibrosis transmembrane conductance regulator (cftr) and Na+/Cl−-taurine transporter (taut) was determined Gapdh expression was used as internal control for normalization of target gene expression Results (mean ± s.e.m.) were from five independent experiments * P < 0.05, ** P < 0.01, *** P < 0.001 vs control subunit α3 (at1α3)], and calcium-transporting ATPase (at2b2) were increased (p < 0.05) The mRNA expression level of V-type proton ATPase (vpp1) was significantly reduced (p < 0.05) while the levels of aquaporin-3 (aqp3) and Na+/K+/2Cl− cotransporter (nkcc, s12a2) mRNA showed no noticeable changes (Fig 2) For the key factors involved in miR biogenesis, the mRNA expression levels of drosha RNase III endonuclease (drosha), exportin-5 (xpo5), dicer ribonuclease III (dicer-1) and argonaute-2 (ago2) in ex-vivo gill filaments were measured at 4, and 24 h of post-hypertonic treatment (Fig 3) In general, the expression levels of drosha, and xpo5 transcripts were significantly reduced under hyperosmotic stress The expression levels of dicer-1, however was significantly increased Hyperosmotic treatment induced differential expression of miRs in ex-vivo gill filament model Gill filaments of eels were ex-vivo cultured in isotonic (300 mOsmol l− 1) or hypertonic (600 mOsmol l− 1) L-15 medium (PS, gentamycin, FBS) at 23 °C for and h Total RNA was extracted and isolated using mirVana (Invitrogen) The A260/A280 value of the isolated RNA was > 1.8, and the RIN was over Library construction was prepared for miR sequencing A total of 568 million quality-trimmed raw reads were obtained from the small RNA sequencing (Additional file 1: ST1) De novo analysis identified 658 and 662 miR precursors from the sequencing samples at and h respectively (Table 1) In consideration of Randford p-value, there were 82 and 84 miR precursors found to be not significant at and h treatment respectively The corrected miR precursors were 576 and 578 at and h treatments Figures 4a, b show the volcanic plots of deregulated miRs of the samples In the h treatment, the expression levels of 81 miR precursors were found to be significantly different (DESeq2 adjusted p-value < 0.05) There were 42 miRs upregulated and 39 miRs downregulated In h treatment, there were 55 differentially expressed miR precursors in which 24 miR precursors were upregulated and Ng et al BMC Genomics (2020) 21:208 Page of 13 Fig Hypertonic stress induces differential gene expression of ion transporting enzyme in gill filament culture Differential gene expression of ATPase Na+/K+-transporting subunit alpha (at1a1), ATPase Na+/K+-transporting Subunit Alpha (at1a3), calcium-transporting ATPase (at2b2), aquaporin (aqp3), V-type proton ATPase (vpp1) and Na+/K+/2Cl−-cotransporter (nkcc) was determined in control (Ctrl: 300 mOsmol l− 1) or hypertonic-treated gill filaments ((Hyper: 600 mOsmol l− 1, 4, 8, 24 h) by qRT-PCR Relative expression level was normalized by gapdh Results (mean ± s.e.m.) were from five independent experiments * P < 0.05, ** P < 0.01 vs control 31 were downregulated (DESeq2 adjusted p < 0.05) To prioritize potential differentially regulated miRs, the significance of p-value was set to p < 0.001 This led to a reduction in the numbers of differentially expressed miRs from 81 to 18 at h, and 79 to 10 at h treatments The Venn diagram (Fig 4c) shows the common differentially expressed miRs at and h The two miRs, miR-29b-3p and miR-200b-3p were commonly downregulated at both time points of the hyperosmotic treatments (Fig 4d) The differentially expressed miRs were validated using realtime PCR of the samples from isotonic and hypertonic treated gill filaments (Fig 4e) Since the suppression of gene expression is one of the major functions of miR, a transcriptome analysis of differential gene expression in ex-vivo gill filament-culture was conducted There were 2085 differentially expressed genes (DEGs) at h of the hypertonic treatment (Fig 5a), including 890 upregulated genes and 1195 downregulated genes (Additional file 1: ST3) In h of hyperosmotic treatment, 1670 DEGs including 841 upregulated genes and 829 downregulated genes were identified (Fig 5b, Additional file 1: ST4) In the comparison of DEGs from the h and h treatments, 577 commonly upregulated and 711 downregulated genes were observed (Fig 5c) Integrated miR-inhibition and Transcriptome analysis in primary gill culture model To underpin the gene targets, specific miR-inhibitors were used to block the activities of miR-29b-3p and miR-200b-3p Figure 6a showed the effects of the inhibition on the individual miRs in primary gill cell culture The inhibition of miR-29b-3p did not affect the expression level of miR-200b-3p, and vice versa Supplementary Tables and showed the list of upregulated gene targets in cells after the treatment with miR-29b-3p and miR-200b-3p inhibitors, respectively We overlaid the transcriptomic data of 4- and h-upregulated genes (ST3 & ST4) and of miR-inhibition, to select two targetgenes [i.e., krueppel-like factor (klf4) and homeobox protein meis2] for miR-29b-3p (Fig 6b), and one targetgene (slc17a5) for miR-200b-3p (Fig 6c) miRanda algorithm was then used to predict the binding of the individual miRs to the target genes TransDecoder (version 5.0.2) was used to determine the coding regions Ng et al BMC Genomics (2020) 21:208 Fig Hypertonic stress limits gene expression of miRNA biogenesis factors in gill filament Gill filaments were subjected to different timepoint of hypertonic treatment in the differential gene expression analysis Expression of miRNA biogenesis key factors, drosha, exportin (xpo5), dicer1 and argonaute (ago2), was determined in control (Ctrl: 300 mOsmol l− 1) or hypertonic-treated (Hyper: 600 mOsmol l− 1) gill filaments for 4, and 24 h by qRT-PCR Gapdh was used as internal control Results (mean ± s.e.m.) were from five independent experiments * P < 0.05, ** P < 0.01, *** P < 0.001 vs control including 5′ UTRs and 3′ UTRs of transcripts [16] The analysis showed that miR-29b-3p and miR-200b-3p could bind to the 3′ untranslated region (3’UTR) of klf4 and slc17a5 (Fig 6d) The binding site of klf4 is at 3’UTR between 447 to 468, while the binding site of s17a5 is at the 3’UTR between 53 to 73 Discussion miRs are a class of endogenous and conserved small RNA molecules (~ 22 nt) that play gene regulatory roles in targeting mRNAs for cleavage or translational repression [17] Based on the finding of the recent studies, miRs are found to have a pivotal role in the regulation of fluid and electrolyte balance [18, 19] It warrants further investigation to identify tonicity-inducible miRs and to determine their potential roles in osmoregulatory responses In the present study, using ex-vivo gill filament culture model, we characterized the hyperosmotic responses with respective to the expression levels of Page of 13 some well-defined hyper- and hypo-osmotic genes (i.e regulators and transporters) The involvement of miRbiogenesis genes and tonicity-responsive miRs were characterized Possible mRNA candidates were predicted using genome-wide analysis of microRNA-mRNA interactome In the first part of the study, we characterized the exvivo gill filament culture model with regard to its responses to hyper-osmotic stress The purpose is to establish a culture model, which retains the three-dimensional organization (primary and secondary lamellae) and takes into account of all the cell types [pavement cells (PVCs), chloride cells (CCs), mucous cells and undifferentiated cells] of gill tissues in experiments In this study, numerous well-characterized hyper-osmotic and hypo-osmotic inducible genes were chosen to evaluate the functional responses and the validity of the culture The hyperosmotic inducible genes, like ostf1 [20, 21], nherf [22], wnk1 [23] are regulatory proteins known to modulate gene transcription, apical trafficking of transmembrane G-protein coupled receptors/ion-transporters and epithelial chloride (Cl−) transport respectively The hyperosmotic ostf1 induction was demonstrated in intact fish and primary gill epithelial cell culture [24] Its expression could also be stimulated by the seawater-adapting hormone, cortisol [25] The orchestrating role of ostf1 to integrate environmental and hormonal signals for osmosensory function was documented In our study, ostf1 mRNA was stimulated in our ex-vivo culture model, under hyperosmotic stress Our data also showed the upregulation of nherf1, suggesting its function in hyperosmotic adaptation Intriguingly, it was reported that nherf1 negatively regulated the apical localization of Na+/H+ exchanger (nhe) in renal brush border cells [26] Nhe facilitates Na+ retention in hypotonic solution, in exchange of H+, the process is important for freshwater adaptation Nhef1 was found to be expressed in ionocytes in zebrafish embryos [12] The downregulation of nherf1 blocked Na+ accumulation in ionocytes Presumably, in our study the upregulation of nherf1 in hyperosmotic gill filaments might downregulate nhe Hyperosmotic acclimation is known to stimulate Na+ and Cl− secretion in ionocytes via the upregulation of kir, Na+/K+-ATPase (at1a1 and at1a3), and cftr The sodium pump generated an extracellular Na+ gradient to facilitate the transcellular co-transport of Cl− to cell cytoplasm, followed by Cl− secretion via apical cftr All these transporters were upregulated in the hyperosmotic treated gill filaments The calcium-transporting ATPase (at2b2) is known to reduce intracellular Ca2+ to maintain calcium homeostasis, which could be perturbed by osmotic stress and seawater adaptation in eels [27, 28] The Na+/K+/ 2Cl−-cotransporter (s12a2) aids in the secondary transport of Na+, K+ and Cl−, driven by Na+-gradient established by Na+/K+-ATPase [29] However, there was no noticeable induction of sl2a2 in this culture model In addition to ion Ng et al BMC Genomics (2020) 21:208 Page of 13 Table Number of dysregulated miRNA caused by hypertonic stress ex vivo approach small RNA seq n = Japanese eel gill filaments 600 mOsmol l-1 h Ctrl vs Hyper 600 mOsmol l-1 h p-value < 0.05 p-value > 0.05 p-value > Total All miRNA precursors 101 512 45 658 Randford p-value (not significant) 20 58 82 Removed miRNA precursor with not significant Randford p-value 658–82 576 p-value < 0.05 p-value > 0.05 p-value > Total Ctrl vs Hyper 600 mOsmol l-1 h 81 454 41 576 up regulated 42 255 19 316 down regulated 39 199 22 260 p-value < 0.05 p-value > 0.05 p-value > Total 600 mOsmol l-1 h Ctrl vs Hyper 600 mOsmol l-1 h All miRNA precursors 65 556 41 662 Randford p-value (not significant) 10 69 84 Removed miRNA precursor with not significant Randford p-value 662–84 578 p-value < 0.05 p-value > 0.05 p-value > Total Ctrl vs Hyper 600 mOsmol l-1 h 55 487 36 578 Up-regulated 24 282 19 325 Down-regulated 31 205 17 253 secretion, hyperosmotic acclimation involves the accumulation of organic osmolytes, like taurine to increase cellular osmolality The expression levels of the taurine transporter, taut was upregulated in the hyperosmotic treated gill filaments The aquaporin-3 (aqp-3) that mediates the translocation of water, glycerol, urea and other small solutes, was highly expressed in freshwater eel gills [30] No significant reduction in its expression levels was measured in this study On the other hand, the transporter (vpp1) involved in hypo-osmotic acclimation, was downregulated in the hyperosmotic treated gill filaments As compared with the primary gill cell model, it is a twodimensional culture and comprises mostly PVCs [31, 32] In the past studies, using primary gill PVC culture under hyperosmotic treatment, upregulation of ostf1, Na+/K+ATPase and taut mRNA expression were detected [31, 33] The stimulation of cftr, kir, and nherf1 expression in the hyperosmotic-treated culture, has not yet been reported Indeed, the expression of cftr and kir are known to be cell-specific, localized in seawater CCs The low percentage of CCs in the primary cell culture made the model not representative as compared with the ex-vivo gill filament culture for studying gill physiology Collectively, our data demonstrated that the ex-vivo gill filament culture exhibited distinctive responses to hyperosmotic acclimation It would be a useful model to investigate underlying mechanisms of osmotic responses in fish gills With hindsight, the involvement of miRs in fluid and electrolyte transport has become apparent A number of studies have illustrated the implications of miRs in ion transport in renal and non-renal tissues, showing a pivotal role of miRs in the regulation of fluid and electrolyte balance In the present study, we studied the expression of four major factors involved in miR biogenesis, using the ex-vivo gill filament culture Briefly, RNA polymerases II and III are involved in primary-miR (primiR) transcription [34, 35] Upon nuclear cleavage of pri-miR by the Drosha RNase III endonuclease, a ~ 6070 nt miRNA precursor (pre-miRNA, a stem loop intermediate) is produced and transported from cell nucleus to cytoplasm in a Ran-GTPase dependent manner by the export receptor Exportin-5 [36, 37] The pre-miRNA is then processed in the cytoplasm by the action of another RNase III endonuclease, Dicer to produce mature miR, followed by Argonaut proteins to target through sequence complementarity in RNA-induced silencing complex [38] In this study, the mRNA expression levels of drosha, xpo5 and dicer1 were dysregulated under hyperosmotic treatment, indicating that the treatment modulated miR-biogenesis in gill cells Since miRs take part in various aspects of physiological functions, including tissue remodeling and cell survival, it is anticipated an alternation of miR expression in the hyperosmotic treated gill filaments Thus, miR sequencing was Ng et al BMC Genomics (2020) 21:208 Page of 13 Fig Expression of miR-29 and miR-200 is dysregulated in gill filament culture under hypertonic stress Gill filaments were challenged in hyperosmotic (Hyper: 600 mOsmol l− 1) media for h and h and were subsequently processed for small RNA sequencing a & b Volcano plot showed differentially expressed miRs under h (a) or h (b) hypertonic treatment in gill filaments compared with the control The p < 0.05 and |Log2 (fold change)| > were set as threshold for significantly differential expression In h hypertonic treatment, 81 differentially expressed miRs including 42 up-regulated miRs and 39 down-regulated miRs were identified, while in h hypertonic treatment, 55 differentially expressed miRs including 24 up-regulated miRs and 31 down-regulated miRs were identified c Venn diagram shows the similarity of differentially expressed miRs under h and h hypertonic treatments Two miRs, miR-29b-3p and miR-200b-3p, with significant expression changes (p < 0.001) in both hypertonic treatment groups were selected for further analysis d Bar charts exhibit the differential expression levels of miR-29b-3p and miR-200b3p in control and hypertonic groups (4 or h) based on deep sequencing data e qRT-PCR was performed to validate the expression pattern of miR-29b-3p and miR-200b-3p snRNA U6 was used as internal control for normalization of miRNA expression Results (mean ± s.e.m.) were from five independent experiments * P < 0.05, ** P < 0.01 vs control conducted using the gill filaments at two consecutive time-points Bioinformatics analysis of the dysregulated miRs from the two time-points at high stringency of filtering (p < 0.001) shortlisted two miR candidates, miR29b-3p and miR-200b-3p In the literatures, the family of miR-29 was found to respond to environmental stress and cellular repairing processes In zebrafish model, the involvement of miR-29 in acute environmental stress (cold stress) was studied [39], in which the miR targeted on a core clock gene per2 to enhance cold tolerance of the fish larvae Moreover, miR-29b was reported to play a role in cell regeneration, associated with the processes of cell survival and cytoskeleton reorganization in zebrafish [40] Furthermore, miR-29 was recognized to ... The expression levels of dicer-1, however was significantly increased Hyperosmotic treatment induced differential expression of miRs in ex- vivo gill filament model Gill filaments of eels were ex- vivo. .. expression of miRNA biogenesis factors in gill filament Gill filaments were subjected to different timepoint of hypertonic treatment in the differential gene expression analysis Expression of miRNA... transporters) The involvement of miRbiogenesis genes and tonicity-responsive miRs were characterized Possible mRNA candidates were predicted using genome- wide analysis of microRNA-mRNA interactome In the